The Betacoronavirus genus includes two viruses, SARS-CoV and MERS-CoV that emerged from animal reservoirs to produce severe pulmonary disease in man in 2002, and 2012, respectively. In this application we propose to study the RNA secondary structure of MERS-CoV and two related coronaviruses, mouse hepatitis virus (MHV) and bovine coronavirus (BCoV) with the goal of identifying and functionally characterizing novel conserved RNA secondary structures in the genomes of these viruses. In aim one we will employ a state-of- the-art, high throughput approach, SHAPE-MaP, to generate biochemical data that allows the creation of highly accurate models of RNA secondary structures of large RNAs, such as coronavirus genomes. Through comparison of the RNA secondary structures of the genomes of MHV, BCoV, and MERS-CoV we will seek previously undiscovered phylogenetically conserved RNA secondary structures that are strong candidates to play a functional role in viral replication. In aim 2 we will employ reverse genetic approaches to characterize the functional role in MHV and MERS-CoV replication of conserved secondary structures identified in aim 1.